sirna Screening for Genes Involved in HSV-1 Replication Samantha J. Griffiths * and Jürgen Haas
|
|
- Ashlie Townsend
- 5 years ago
- Views:
Transcription
1 sirna Screening for Genes Involved in HSV-1 Replication Samantha J. Griffiths * and Jürgen Haas Division of Pathway and Infection Medicine, University of Edinburgh, Edinburgh, UK *For correspondence: samantha.griffiths@ed.ac.uk [Abstract] Small interfering RNAs (sirnas) are small (typically nucleotides) RNA molecules capable of silencing gene expression post-transcriptionally and as such, they provide a simple method by which the role of individual genes in complex cellular systems can be easily assessed. As sirnas are easy to use experimentally, and can be designed to target any gene (including pathogens), their use is perfectly suited to and easily adapted to high-throughput genome-wide screening methodologies and a range of phenotypic assays. Here we describe the use of a large sirna library (>8,000 genes targeted individually) to screen for and identify host factors functionally involved in the replication of a human herpesvirus (Herpes simplex virus type 1; HSV-1) (Griffiths et al., 2013; Griffiths, 2013). Materials and Reagents 1. Hela cells (ATCC, catalog number: CCL-2) 2. sirna library (e.g. human sigenome SMARTpool Druggable Genome sirna library 0.5 nmol) (Thermo Fisher Scientific, catalog number: G ) 3. 5x sirna buffer (Thermo Fisher Scientific, catalog number: B UB-100) 4. RISC-free (RSCF) control sirna (20 nmol) (Thermo Fisher Scientific, catalog number: D ) 5. Scrambled, non-targeting control sirna (20 nmol, human) (Thermo Fisher Scientific, catalog number: D ) 6. Assay-specific control sirnas 7. Fetal bovine serum (FBS) (LabTech, catalog number: FCS-SA-10454) 8. Penicillin:streptomycin (5,000 units/ml each) (Lonza, catalog number: DE17-603E) 9. 1x trypsin-edta liquid (0.05% Trypsin, 0.53 mm EDTA-4Na) (Life Technologies, catalog number: ) 10. Phosphate buffered saline without magnesium or calcium (Lonza, catalog number: F) 11. DMEM 4.5 g glucose/litre with L-glutamine and sodium pyruvate (Lonza, catalog number: BE12-604F) Copyright 2014 The Authors; exclusive licensee Bio-protocol LLC. 1
2 12. 1x DMEM/F12 1:1 (15 mm HEPES + L-glutamine, no phenol red) (Life Technologies, catalog number: ) 13. Hank s balanced salt solution (HBSS) (Thermo Fisher Scientific, catalog number: HYC W) 14. Dharmafect 1 sirna transfection reagent (Thermo Fisher Scientific, catalog number: T- 2001) 15. Sterile DNase and RNase-free distilled water (Life Technologies, catalog number: ) 16. Ethanol (diluted in sterile RNase and DNase-free water) 17. CellTiter-Blue cell viability reagent (Promega Corporation, catalog number: G8081) 18. HSV-1-eGFP reporter virus (Arthur et al., 2001) 19. Growth medium (see Recipes) 20. Transfection medium (see Recipes) Equipment 1. Pipette tips for robotic liquid handler (Axygen Zymark 200 µl sterile tips) (VWR International, catalog number: AXYGZT-200-L-R-S) 2. Polystyrene plate covers for 96-well plates (Thermo Fisher Scientific, catalog number: AB0752) 3. Aluminium heat-sealing film for storage microplates (Thermo Fisher Scientific, catalog number: AB-0559) 4. Adhesive plate seals (Starlab, catalog number: E ) well storage plates (Thermo Fisher Scientific, catalog number: AB-0781) 6. Black 384-well clear flat-bottomed, sterile, tissue culture-treated plates (VWR International, catalog number: ) cm 2 filter cap tissue culture flasks (Sigma-Aldrich, catalog number: C7106) cm 2 filter cap tissue culture flasks (Sigma-Aldrich, catalog number: C7481) ml centrifuge tube (Corning, product number: ) 10. Disposable hemocytometer (KOVA Glasstic slide 10 with counting grid) (HYCOR Biomedical, catalog number: 87144E) 11. Class II Microbiological safety cabinet 12. Humidified cell culture incubator (37 C, 5% CO2) 13. Pipette aid (Alpha laboratories, catalog number: E) 14. Centrifuge (e.g. Eppendorf, catalog number: ) 15. Light microscope Copyright 2014 The Authors; exclusive licensee Bio-protocol LLC. 2
3 16. 8-channel multichannel pipette (2-10 μl volume) (Thermo Fisher Scientific, catalog number: ) 17. Multidrop 384 reagent dispenser (Thermo Fisher Scientific, catalog number: ) 18. Multidrop 384 standard tube dispensing cassette (Thermo Fisher Scientific, catalog number: ) 19. RapidPlate 384 robotic liquid handler (or equivalent) (discontinued) (QIAGEN) 20. Microplate heat sealer (e.g. ALPS 50 V Microplate heat sealer) (Thermo Fisher Scientific, catalog number: AB-1443) 21. Gas-permeable adhesive microplate seals (Thermo Fisher Scientific, catalog number: AB-0718) 22. Barcode printer (e.g. Brady LABXPERT printer) (Sigma-Aldrich, catalog number: Z664588) 23. Brady LABXPERT printer labels (Thermo Fisher Scientific, catalog number: ) 24. Fluorescent plate reader (e.g. POLARstar OPTIMA, BMG LABTECH) 25. Stacker for plate reader (BMG LABTECH) Software 1. Microsoft Excel Procedure 1. Remove the lyophilised sirna library (76 x 96-well plates) from storage at -80 C and equilibrate to room temperature. 2. Pellet well contents by centrifuging for 10 min at 1,000 rpm and replace plate seals with clean plastic lids. 3. Clean a Multidrop 384 dispensing cassette with 3 x 25 ml 70% ethanol and rinse with 3 x 50 ml sterile DNase and RNase-free water. 4. Prepare a sufficient volume of 1x sirna buffer by diluting 5x sirna buffer in sterile DNase and RNase-free water. 5. Resuspend the 0.5 nmol sirna library to 3 µm by dispensing 165 µl 1x sirna buffer to columns 3-12 of the sirna library plates (96-well plates) (see Note 1; Figure 1A). 6. Using a label maker, print text or barcode labels for 384-well Master Plates (see Note 2). 7. Adhere labels to 384-well storage plates (for a library of 76 x 96-well plates you will need 19 x 384-well storage plates and labels). These will be the 384-well Master Plates. Copyright 2014 The Authors; exclusive licensee Bio-protocol LLC. 3
4 8. Clean a dispensing cassette for the Multidrop 384, as above, and dispense 110 µl 1x sirna buffer into columns 1 and 2 of the 384-well Master plate (see Note 3; Figure 1B). Figure and 384-well plate preparation 9. Dilute the assay-specific sirna controls to 300 nm in 1x sirna buffer (see Note 4). 10. Manually transfer 110 µl of each sirna control, in horizontal duplicates, to columns 3 and 4 in every 384-well Master Plate i.e. transfer RSCF sirna to wells A3 and A4; transfer non-targeting sirna to wells B3 and B4 etc. (Figure 1C). 11. Dispense 100 µl 1x sirna buffer to columns 5-24 in each 384-well master plate using a Multidrop Using a robotic liquid handler (such as the QIAGEN RapidPlate 384), with an individual box of Zymark robotic tips for each sirna 96-well Library Plate, transfer 11 µl from the sirna library plates (3 µm sirna) to the 384-well Master plate. Each 384-well plate takes 4 x 96-well sirna Library plates (Figure 1D). These plates can be stored at -80 C until needed. 13. Use a microplate heat sealer and aluminium heat seals to seal the sirna library plates before transferring to a -80 C for long-term storage. Copyright 2014 The Authors; exclusive licensee Bio-protocol LLC. 4
5 14. Print appropriate barcode or text labels for the 384-well assay plates containing relevant information (replicate number, assay type etc.) (see Note 5). 15. Stick labels to black, tissue-culture treated, flat, clear-bottomed 384-well plates. 16. If frozen, thaw the 384-well Master Plates and briefly centrifuge for 1 min at 1,000 rpm before transferring 10 µl to three replicate assay plates using a robotic liquid handler and an individual box of Zymark robotic tips for each quadrant of the 384-well plate. 17. Cover prepared assay plates with an adhesive plate seal, place in sealable bags and stored at -80 C for up to 2 weeks before use (see Note 6). 18. Calculate the number of low passage Hela cells in a confluent T175 flask, and the total number of cells required for your assay. Based on these numbers determine the number of 50% confluent T175 flasks required to provide the total number of cells for your assay. 19. Several days before your assay, prepare Hela cells such that there will be sufficient 100% confluent T175 flasks the day before the sirna transfection. 20. The day before transfection, remove medium from the confluent T715 flasks of Hela cells, and rinse cells with warmed sterile phosphate buffered saline. 21. Discard wash, and dislodge cells by incubating for around 5 min at 37 C in 5 ml prewarmed trypsin. 22. Inactivate trypsin by adding 10 ml growth medium and transfer the pre-determined volume of cells to sufficient T175 flasks such that they will be 50% confluent the following day (see Note 7). 23. The next day, thaw the replicate assay plates and centrifuge for 5 min at 1,000 rpm. Remove the plate seals and cover with lids. 24. Dilute the Dharmafect 1 (DF1) transfection reagent to 0.6% in the appropriate volume of HBSS and add 10 µl to each well of the 384-well assay plates using a dispensing cassette for the Multidrop Centrifuge the assay plates for 1 min at 1,000 rpm and incubate at room temperature for 20 min for transfection complexes to form. 26. Remove growth medium from the Hela cells, rinse in PBS and dislodge cells by incubating for ~5 min at 37 C in 5 ml trypsin. 27. Inactivate the trypsin by adding 10 ml of transfection medium. 28. Mix each flask well and pool together all cells before mixing well and removing 5 x 12 μl aliquots of cell suspension for counting in a disposable hemocytometer. 29. Calculate the average cell density and dilute to 7.5 x 10 4 cells/ml. 30. After the 20 min incubation time, transfer 40 µl cells to every well in the assay plates and incubate plate in a humidified incubator at 37 C for 48 h to ensure gene depletion prior to investigating your assay output. 31. After 48 h, determine the effect of gene depletion on cell viability. Copyright 2014 The Authors; exclusive licensee Bio-protocol LLC. 5
6 32. Thaw the CellTitre Blue reagent (or equivalent) and add 5 µl per well of each Cell Viability replicate plate using the Multidrop Return the plates to the 37 C incubator for 2 h before measuring the fluorescence signal (485 nm) of each plate using a POLARstar OPTIMA plate reader (or equivalent). The effect of sirna depletion on cell viability is determined by comparing fluorescence to the average fluorescence of mock-transfected cells (columns 1 and 2) (see Note 8). 34. To infect the duplicate assay plates with HSV-1-eGFP, thaw thehsv-1-egfp virus stock in a 37 C water bath until just thawed, before diluting to a multiplicity of infection (MOI) of 0.5 in phenol red-free DMEM: F12/15 mm HEPES/L-glutamine/5% FCS/pen-strep (see Note 9). 35. Remove media from all plates by inverting and shaking over a suitable liquid waste container. 36. Add 10 µl phenol red-free growth medium to columns 1 and 2 of every plate manually using a low volume (2-10 µl) 8-channel multichannel pipette (see Note 10). 37. Add 10 µl diluted virus to the remaining wells (columns 3-24) using a Multidrop 384 and centrifuge for 2 min at 1,000 rpm to remove air bubbles and ensure the cell monolayer is covered by the small volume of inoculum. 38. Transfer infection assay plates to the 37 C incubator for 1 h to allow virus to adsorb before adding 50 µl phenol red-free growth medium to every well using the Multidrop Cover the plates with a gas-permeable adhesive seal, stack and cover the top plate with a plastic lid before returning all assay plates to the incubator for 20 h. 40. Place assay plates in the stacker equipment of a fluorescence plate reader and monitor virus replication as a measure of GFP fluorescence in a fluorescent plate reader every 3-4 h until replication plateaus (see Note 11). 41. Once the virus replication cycle has completed, calculate the replication slope over the linear phase of growth for each well and determine the effect of gene depletion on virus replication by comparing the replication slope to the average replication slope of control wells (mock, RSCF and non-targeting sirna-transfected) on a per plate basis. The LINEST equation in Microsfot Excel can be used to calculate replication slopes. This formula utilises the known x values (the hours post-infection) and the known y values (the relative fluorescence at each time post-infection). Firstly, generate a table with the Hours post-infection and the relative fluorescence of your samples (Figure 1A). Plot these data and identify the linear phase of replication (Figure 1B; red line indicates linear phase). Use the linest formula to calculate the replication slope, and normalise the slope of each condition to that of your control 9in this example, normalise to Mock transfected cells; Figure 1C-D). Copyright 2014 The Authors; exclusive licensee Bio-protocol LLC. 6
7 Figure 2. Calculation of replication slopes Notes 1. Library plates are diluted 1: 10 to 300 nm when generating the 384-well Master Plates, and diluted a further 1: 6 during the assay. This gives a final sirna concentration of 50 nm. If a higher/lower sirna concentration is preferred, adjust the resuspension volumes accordingly. Columns 1 and 2 of the 96-well sirna Library plates are empty to enable the user to include assay-specific controls. 2. As sirnas are temperature sensitive, we recommend the generation of a 384-well master plate which contains sufficient sirna to complete three full screens in triplicates. sirna can be dispensed into replicate assay plates at the same time to ensure an equal number of freeze-thaw cycles of assay plates and reproducible assay results. 3. The addition of sirna buffer alone to columns 1 and 2 of each 384-well master plate will result in untransfected control cells. For our virus replication assay, these will remain uninfected. 4. Be sure to include mock-transfected cells (transfection reagent alone), sirna controls (such as RISC-free and a scrambled, non-targeting sirna) and assay-specific sirnas which display a known range of phenotypes (strong positive effect, moderate effect, strong negative effect). Copyright 2014 The Authors; exclusive licensee Bio-protocol LLC. 7
8 5. For a virus replication assay we recommend carrying out each assay in triplicate. One replicate should be used for a cell viability assay (as virus will not replicate if sirna gene depletion has a cytotoxic effect) and the virus replication assay is done in duplicates. 6. Whilst diluted sirna will be stable for some time at -80 C the risk of evaporation of such a small volume is high. We would recommend storing assay plates for no longer than 2 weeks before use. 7. sirna transfection and virus replication is optimal when cells were sub-confluent (~50%) and freshly passaged prior to use. 8. The Cell Titre Blue reagent is metabolised to produce a fluorescent signal. The cell viability is therefore demonstrated by the rate of metabolism of the cells. The effect of gene depletion on cell viability is determined by comparing the fluorescence of sirnatransfected wells to the average fluorescence of control, non-transfected wells (columns 1 and 2) on a per plate basis to avoid inter-plate variations. Normalising the fluorescence in this way enables identification of cytotoxic sirnas as well as those increase cell proliferation. As per manufacturer s instructions, a fluorescence value of <70% of the control cells is considered cytotoxic. Cytotoxic sirnas are excluded from subsequent data analyses. 9. It is important to infect cells with a known quantity of virus (multiplicity of infection; MOI) to generate a suitable growth curve. We find a multiplicity of infection (MOI) of 0.5 [0.5 virus plaque forming units (PFU) per cell] gives an ideal kinetic growth curve from 20 to 80 h post-infection. The MOI is dependent upon the cell number and volume used for the infection, so the appropriate dilution factor should be calculated based upon the starting titre of your virus stock. For example, to infect 3 x 10 3 cells at MOI 0.5 in 10 μl we need to have a virus preparation containing 1.5 x 10 5 PFU/ml. A starting stock of 6 x 10 7 PFU/ml should therefore be diluted 1 in Tissue culture plasticware, cells and growth medium all have some level of fluorescence. When utilising fluorescent reporter genes it is therefore essential to have appropriate controls to provide background fluorescence readings. For this assay, the fluorescence from uninfected cells is used as background. 11. Replication is monitored over regular intervals to enable a complete growth curve (fluorescence over time) to be plotted. When comparing HSV replication between untreated and treated samples we use the slope of replication over the linear growth phase. It is therefore important to have as many measurements over this phase as possible (with a minimum of 6 for statistical reliability of the slope calculation). A test replication assay with your own equipment will allow the time of linear growth to be established, and assay timings can be adjusted to ensure measurements can be taken over this period. Copyright 2014 The Authors; exclusive licensee Bio-protocol LLC. 8
9 Recipes 1. Growth medium DMEM 5% FCS 1% pen-strep 2. Transfection medium Phenol red-free DMEM: F12 15 mm HEPES L-glutamine 5 % FCS Acknowledgments This protocol has been edited from that described in Griffiths et al. (2013) PLOS Pathogens. The authors are grateful to Prof. Peter Ghazal for provision of the sirna libraries, and Marie Craigon for technical assistance with library handling, management and establishment of protocols. The authors are grateful to the MRC (G J.H.), BBSRC (BB/B019621/1 P.G.), Scottish Enterprise (P.G.) and the Wellcome Trust (WT P.G.) for funding. References 1. Arthur, J. L., Scarpini, C. G., Connor, V., Lachmann, R. H., Tolkovsky, A. M. and Efstathiou, S. (2001). Herpes simplex virus type 1 promoter activity during latency establishment, maintenance, and reactivation in primary dorsal root neurons in vitro. J Virol 75(8): Griffiths, S. J., Koegl, M., Boutell, C., Zenner, H. L., Crump, C. M., Pica, F., Gonzalez, O., Friedel, C. C., Barry, G., Martin, K., Craigon, M. H., Chen, R., Kaza, L. N., Fossum, E., Fazakerley, J. K., Efstathiou, S., Volpi, A., Zimmer, R., Ghazal, P. and Haas, J. (2013). A systematic analysis of host factors reveals a Med23-interferon-lambda regulatory axis against herpes simplex virus type 1 replication. PLoS Pathog 9(8): e Griffiths, S. J. (2013). Screening for host proteins with pro- and anti-viral activity using high-throughput RNAi in Virus-Host Interactions. In: Methods in Molecular Biology. Vol. 1064: Copyright 2014 The Authors; exclusive licensee Bio-protocol LLC. 9
VZV Replication Assays Samantha J. Griffiths * and Jürgen Haas
VZV Replication Assays Samantha J. Griffiths * and Jürgen Haas Division of Pathway and Infection Medicine, University of Edinburgh, Edinburgh, UK *For correspondence: samantha.griffiths@ed.ac.uk [Abstract]
More informationMeasuring cell proliferation using the CyQUANT Cell Proliferation Assay with SpectraMax Microplate Readers
APPLICATION NOTE Measuring cell proliferation using the CyQUANT Cell Proliferation Assay with SpectraMax Microplate Readers Introduction Quantitation of cell proliferation using fluorescence allows one
More informationHIV-1 Pseudovirus Neutralization Protocol using Ghost cells
HIV-1 Pseudovirus Neutralization Protocol using Ghost cells Institute of Tropical Medicine, Belgium Neutnet code: 6A Contact person: Leo Heyndrickx; LHeyndrickx@itg.be 1. AIM: This protocol describes the
More informationUsing the xcelligence RTCA SP Instrument to Perform GPCR Assays
xcelligence Real-Time Cell Analysis GPCR Assay Protocol Using the xcelligence RTCA SP Instrument to Perform GPCR Assays Overview The xcelligence RTCA SP Instrument The xcelligence Real-Time Cell Analyzer
More informationUsing the xcelligence RTCA SP Instrument to Perform Cytotoxicity Assays
xcelligence Real-Time Cell Analysis Cytotoxicity Assay Protocol Using the xcelligence RTCA SP Instrument to Perform Cytotoxicity Assays Overview The xcelligence RTCA SP Instrument The xcelligence Real-Time
More informationEvolution of Escherichia coli to Macrophage Cell Line Migla Miskinyte and Isabel Gordo *
Evolution of Escherichia coli to Macrophage Cell Line Migla Miskinyte and Isabel Gordo * Evolutionary Biology Group, Instituto Gulbenkian de Ciência, Oeiras, Portugal *For correspondence: igordo@igc.gulbenkian.pt
More informationAlt-R CRISPR-Cpf1 System:
user guide Alt-R CRISPR-Cpf1 System: Delivery of ribonucleoprotein complexes in HEK-293 cells using the Amaxa Nucleofector System See what more we can do for you at www.idtdna.com. For Research Use Only
More informationRatiometric Calcium Assay Kit
BD Technical Data Sheet Ratiometric Calcium Assay Kit Product Information Catalog Number: 644244 Components: Ratiometric Calcium Indicator, 10 vials, lyophilized 10X Signal Enhancer, 100 ml Description
More informationPropagation of H7 hesc From: UW (John Stamatoyannopoulos) ENCODE group Date: 12/17/2009 Prepared By: S. Paige/S. Hansen (UW)
Propagation of H7 hesc From: UW (John Stamatoyannopoulos) ENCODE group Date: 12/17/2009 Prepared By: S. Paige/S. Hansen (UW) Growth and Harvest Modifications Addendum to: Propagation of H7 hesc from UW
More informationBD Ratiometric Calcium Assay Kit
BD Technical Data Sheet BD Ratiometric Calcium Assay Kit Product Information Catalog Number: 644243 Components: Ratiometric Calcium Indicator, 1 vial, lyophilized 10X Signal Enhancer, 10 ml Calcium Assay
More informationSTANDARD OPERATING PROCEDURE
STANDARD OPERATING PROCEDURE Culture of the Rat Macrophage Cell Line, NR8383 SOP number: WP1/001 Protocol prepared by: Ewelina Hoffman Developed under NC3R project: NC/C013203/1 INTRODUCTION Procedure
More informationAmaxa Cell Line Nucleofector Kit L
Amaxa Cell Line Nucleofector Kit L For 3T3-L1 (adipocytes) [ATCC CL-173, cryopreserved] Mouse embryonal fibroblast, differentiated into adipocytes; Fibroblast-like cells before differentiation; adipocyte-like
More informationIncuCyte S3 Neuronal Activity Assay
IncuCyte S3 Neuronal Activity Assay For the kinetic quantification of neuronal activity and functional connectivity This protocol provides an overview of the methodology which uses the lentiviral based
More informationSTANDARD OPERATING PROCEDURE
STANDARD OPERATING PROCEDURE Culture of the Human Monocyte-Macrophage Cell Line; U937 SOP number: WP1/002 Protocol prepared by: Ewelina Hoffman & Abhinav Kumar Developed under NC3R project: NC/C013203/1
More informationIncuCyte S3 Neuronal Activity Assay
IncuCyte S3 Neuronal Activity Assay For the kinetic quantification of neuronal activity and functional connectivity This protocol provides an overview of the methodology which uses the lentiviral based
More informationFitness Measurements of Evolved Escherichia coli Migla Miskinyte and Isabel Gordo *
Fitness Measurements of Evolved Escherichia coli Migla Miskinyte and Isabel Gordo * Evolutionary Biology Group, Instituto Gulbenkian de Ciência, Oeiras, Portugal *For correspondence: igordo@igc.gulbenkian.pt
More informationProtocol. High-throughput Transfection Protocol for GoClone Reporter Assays. Tech support: Luciferase Assay System
Luciferase Assay System Protocol High-throughput Transfection Protocol for GoClone Reporter Assays LightSwitch Luciferase Assay System GoClone Reporter Assay Workflow promoter luciferase Step 1: Simultaneously
More informationHuman Skeletal Muscle Myoblast Care Manual: Maintenance and Differentiation from Myoblasts to Myocytes
Human Skeletal Muscle Myoblast Care Manual: Maintenance and Differentiation from Myoblasts to Myocytes STORAGE CONDITIONS Media: Short Term 4 C 6 months -20 C F or research use only. Not approved for human
More informationSupplementary Note 1. Materials and Methods for the BRET procedure. Solutions and reagents
Supplementary Note 1 Materials and Methods for the BRET procedure Solutions and reagents PEI (1 mg/ml): The polyethyleneimine (PEI) powder (Polysciences, Inc.; MW ~25 KDa; cat#: 23966) is dissolved in
More informationWOOD CELL PASSAGING PROTOCOL OVERVIEW
CELL MODEL NAME: Wood Derived from an infiltrating ductal and lobular carcinoma of the breast. Product Category: Cellini Breast Models Catalog #: CB040-000001 Record your lot number here: Please refer
More informationPD-1 [Biotinylated] : PD-L1 Inhibitor Screening ELISA Assay Pair
PD-1 [Biotinylated] : PD-L1 Inhibitor Screening ELISA Assay Pair Pack Size: 96 tests / 480 tests Catalog Number:EP-101 IMPORTANT: Please carefully read this manual before performing your experiment. For
More informationHuman Alanine Aminotransferase (ALT) ELISA Kit. For Reference Only
Human Alanine Aminotransferase (ALT) ELISA Kit Catalog No.: abx572210 Size: 96T Range: 0.312 ng/ml - 20 ng/ml Sensitivity: < 0.132 ng/ml Storage: Store standard, detection reagent A, detection reagent
More informationStandard Operating Procedure
Title: Standard Operating Procedure Flow cytometry-based invasion phenotyping assay Authors: Michel Theron, Richard Hesketh, Sathish Subramanian & Julian Rayner Date: 14.09.2010 All procedures should be
More informationGrowth and Maintenance of the 293A Cell Line
Growth and Maintenance of the 293A Cell Line USER GUIDE Catalog Number R705-07 Publication Number MAN0000303 Revision A.0 For Research Use Only. Not for use in diagnostic procedures. The information in
More informationReprogramming of Murine Somatic Cells Using mir302/367 lentivirus Modification by: Frederick Anokye-Danso, Ph.D
Reprogramming of Murine Somatic Cells Using mir302/367 lentivirus Modification by: Frederick Anokye-Danso, Ph.D MATERIALS Media Sodium Pyruvate (Mediatech; Cat# MT25-000-CI) Leukemia Inhibitory Factor
More informationReproRNA -OKSGM is a non-integrating, self-replicating RNA-based reprogramming vector for generating induced pluripotent stem (ips)
Kit for generating ips cells using ReproRNA -OKSGM, a non-integrating, self-replicating RNA reprogramming vector Product Description ReproRNA -OKSGM is a non-integrating, self-replicating RNA-based reprogramming
More informationCell-Free Protein Expression Kit
Cell-Free Protein Expression Kit Handbook Version v.01, January 2018 Cat# 507024 (Sigma 70 Master Mix Kit, 24 Rxns) Cat# 507096 (Sigma 70 Master Mix Kit, 96 Rxns) Please refer to this product in your publication
More informationVDL300.3 PRODUCTION OF RETROVIRAL VECTOR BY TRANSIENT TRANSFECTION
1. Purpose 1.1. The purpose of this protocol is to produce retrovirus from transfected plasmid DNA. 1.2. This procedure is routinely performed in the Vector Development Laboratory (VDL) following Good
More informationMinimal Bactericidal Concentration for Biofilms (MBC-B) Nicole Billings and Katharina Ribbeck *
Minimal Bactericidal Concentration for Biofilms (MBC-B) Nicole Billings and Katharina Ribbeck * Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA *For correspondence:
More informationProteasome Activity Fluorometric Assay Kit II (Cat. # J4120)
Proteasome Activity Fluorometric Assay Kit II (Cat. # J4120) Each supplied substrate is sufficient for use in 250 X 100 µl reactions to monitor the chymotrypsin-like (Suc-LLVY- AMC), trypsin-like (Boc-LRR-AMC)
More informationHuman Dermal Fibroblast Manual
Human Dermal Fibroblast Manual INSTRUCTIONAL MANUAL ZBM0023.04 SHIPPING CONDITIONS Human Adult or Neonatal Dermal Fibroblast Cells Orders are delivered via Federal Express courier. All US and Canada orders
More informationClonetics Skeletal Muscle Myoblast Cell Systems HSMM Instructions for Use
Lonza Walkersville, Inc. Walkersville, MD 21793-0127 USA U.S. Scientific Support: 800 521 0390 scientific.support@lonza.com EU/ROW Scientific Support: +32 87 321 611 scientific.support.eu@lonza.com Document
More informationMurine in vivo CD8 + T Cell Killing Assay Myoungjoo V. Kim 1*, Weiming Ouyang 2, Will Liao 3, Michael Q. Zhang 4 and Ming O. Li 5
Murine in vivo CD8 + T Cell Killing Assay Myoungjoo V. Kim 1*, Weiming Ouyang 2, Will Liao 3, Michael Q. Zhang 4 and Ming O. Li 5 1 Department of Immunobiology, Yale University School of Medicine, New
More informationab CytoPainter Cell Plasma Membrane Staining Kit Deep Red Fluorescence
Version 1 Last updated 27 April 2017 ab219942 CytoPainter Cell Plasma Membrane Staining Kit Deep Red Fluorescence For staining cell plasma membrane in live cells using our proprietary deep red fluorescence
More informationCAROUSEL CELL PASSAGING PROTOCOL OVERVIEW
CELL MODEL NAME: Carousel Derived from an endometrioid adenocarcinoma of the ovary. Record your lot number here: Product Category: Raphael Ovarian Models Catalog #: AMS.CB010-000001 Please refer to Certificate
More informationaxion Protocol Cell Culture on Microelectrode Arrays Cell Type: ArunA Biomedical - mmngfp+ Mouse Motor Neurons GFP+ BioSystems v. 1.
axion BioSystems Cell Culture on Microelectrode Arrays Cell Type: ArunA Biomedical - mmngfp+ Mouse Motor Neurons GFP+ Protocol v. 1.1 mmngfp+ Mouse Motor Neurons GFP+ from ArunA Biomedical axion BioSystems
More informationNF-κB/Jurkat/GFP Transcriptional Reporter Cell Line
NF-κB/Jurkat/GFP Transcriptional Reporter Cell Line User Manual Store vial in vapor phase of liquid nitrogen on receipt NF-κB/Jurkat/GFP Reporter Cell Line Contents I. Introduction and Background A. Overview
More information3T3-L1 Differentiation Protocol
3T3-L1 Differentiation Protocol Written by Eisuke Kato on 2013/10/09 MATERIALS Dulbecco's Modified Eagles Medium (D-MEM) (High Glucose) with L-Glutamine and Phenol Red High glucose (Wako Chem 044-29765,
More informationCARE MANUAL INSTRUCTIONAL MANUAL ZBM SHIPPING CONDITIONS STORAGE CONDITIONS ORDERING INFORMATION AND TECHNICAL SERVICES
HUMAN ADULT DERMAL FIBROBLAST CARE MANUAL INSTRUCTIONAL MANUAL ZBM0023.01 SHIPPING CONDITIONS Human Adult Dermal Fibroblast Cells Orders are delivered via Federal Express courier. All US and Canada orders
More informationProtocol. GoClone Reporter Constructs: Sample Protocol for Adherent Cells. Tech support: Luciferase Assay System
Luciferase Assay System Protocol GoClone Reporter Constructs: Sample Protocol for Adherent Cells LightSwitch Luciferase Assay System GoClone Reporter Assay Workflow Step 1: Seed cells in plate format.
More informationab Glucose Uptake Assay Kit (Cell-based)
ab204702 Glucose Uptake Assay Kit (Cell-based) Instructions for Use For measuring glucose uptake via flow cytometry and fluorescent microscopy. This product is for research use only and is not intended
More informationQuantiSir General Gene Knockdown Quantification Kit
QuantiSir General Gene Knockdown Quantification Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The QuantiSir General Gene Knockdown Quantification Kit is suitable for quantifying gene
More informationOPPF-UK Standard Protocols: Mammalian Expression
OPPF-UK Standard Protocols: Mammalian Expression Joanne Nettleship joanne@strubi.ox.ac.uk Table of Contents 1. Materials... 3 2. Cell Maintenance... 4 3. 24-Well Transient Expression Screen... 5 4. DNA
More information3T3-L1 Cell Care Manual Maintenance and Differentiation of 3T3-L1 Preadipocytes to Adipocytes
3T3-L1 Cell Care Manual Maintenance and Differentiation of 3T3-L1 Preadipocytes to Adipocytes INSTRUCTION MANUAL SHIPPING CONDITIONS ZBM0009.02 Must be processed upon shipment receipt. STORAGE CONDITIONS
More informationMouse Embryonic Stem Cell Differentiation to Hematopoietic Precursors Hogune Im
Mouse Embryonic Stem Cell Differentiation to Hematopoietic Precursors Hogune Im Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical School, Madison,
More informationTotal Histone H3 Acetylation Detection Fast Kit (Fluorometric)
Total Histone H3 Acetylation Detection Fast Kit (Fluorometric) Catalog Number KA1539 48 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use...
More informationCells and Tissue DNA Isolation 96-Well Kit (Magnetic Bead System) Product # 62500
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Cells and Tissue DNA Isolation 96-Well Kit (Magnetic Bead System)
More informationTACS MTT Assays. Cell Proliferation and Viability Assays. Catalog Number: TA tests. Catalog Number: TA tests
TACS MTT Assays Cell Proliferation and Viability Assays Catalog Number: TA5355-2500 tests Catalog Number: TA5412-5000 tests This package insert must be read in its entirety before using this product. FOR
More informationVDL602.2 RAPID ASSAY FOR DETERMINING ADENOVIRAL VECTOR TITER
1. Purpose 1.1. The purpose of this protocol is to determine the number of infectious adenoviral particles. 1.2. The starting material is purified adenoviral vectors. 1.3. This protocol is based upon the
More informationCalcium Assay Kit. Technical Data Sheet. Product Information. Description. Storage. Materials not included
BD Technical Data Sheet Calcium Assay Kit Product Information Catalog Number: 640176 Size Reagents for 10 plates Components: Calcium Indicator, 1 vial, lyophilized 10X Signal Enhancer, 10 ml 1X Calcium
More informationTranswell Co-culture of Bone Marrow Macrophages with Tumor Cells Mackenzie K. Herroon and Izabela Podgorski *
Transwell Co-culture of Bone Marrow Macrophages with Tumor Cells Mackenzie K. Herroon and Izabela Podgorski * Department of Pharmacology, Karmanos Cancer Institute, Wayne State University School of Medicine,
More informationVDL102.3 Production of Adenovirus in 293 Cells
1. Purpose CENTER FOR CELL & GENE THERAPY 1.1. The purpose of this protocol is to produce adenoviral vectors from transfected plasmid DNA. 1.2. This procedure is routinely performed in the Vector Development
More informationab MTT Cell Proliferation Assay Kit
Version 1 Last updated 22 March 2018 ab211091 MTT Cell Proliferation Assay Kit For the measurement of cell proliferation in cultured cells. This product is for research use only and is not intended for
More informationMaintenance of Novus Cerebral Cortical Neurons
Maintenance of Novus Cerebral Cortical Neurons Catalog nos. NBP2-31284, NBP2-31285, NBP2-31287 thru NBP2-31289, NBP2-31283, NBP2-31295 thru NBP2-31298, NBP2-31344, NBP2-31350 thru NBP2-31353, NBP2-31355
More informationSample Preparation Demonstrated Protocol
10x Genomics Sample Preparation Demonstrated Protocol Fresh Frozen Human Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing NOTICES Notices Manual Part Number CG00039 Legal Notices Rev B
More information3D Mammary Colony-Forming Cell Assay Giusy Tornillo 1* and Sara Cabodi 2
3D Mammary Colony-Forming Cell Assay Giusy Tornillo 1* and Sara Cabodi 2 1 Cardiff School of Biosciences, European Cancer Stem Cell Research Institute, Cardiff University, Cardiff, UK; 2 Department of
More informationHuman Adult Mammary Fibroblast Care Manual
Human Adult Mammary Fibroblast Care Manual INSTRUCTIONAL MANUAL ZBM0062.05 SHIPPING CONDITIONS Human Adult Mammary Fibroblast Cells Orders are delivered via Federal Express courier. All US and Canada orders
More informationSample Preparation Demonstrated Protocol
10x Genomics Sample Preparation Demonstrated Protocol Removal of Dead Cells from Single Cell Suspensions for Single Cell RNA Sequencing NOTICES Notices Manual Part Number CG000093 Legal Notices Rev B 2017
More informationMiraCell Endothelial Cells (from ChiPSC12) Kit
Cat. # Y50055 For Research Use MiraCell Endothelial Cells (from ChiPSC12) Kit Product Manual Table of Contents I. Description...3 II. III. IV. Components...4 Storage...4 Preparation Before Use...4 V. Protocol...5
More informationHuman IFN-α (Interferon-Alpha) Pre-Coated ELISA Kit
Human IFN-α (Interferon-Alpha) Pre-Coated ELISA Kit Catalog No: 90-2235 1 96 well Format (96 tests) Detection Range: 15.6 1000 pg/ml Sensitivity:
More informationJPMA 06, Dec., Case Studies of Established Conditions in Analytical Procedures. Biopharmaceutical Committee Technology Working Committee
CMC Strategy Forum Japan 2016 Case Studies of Established Conditions in Analytical Procedures Biopharmaceutical Committee Technology Working Committee JPMA 06, Dec., 2016 Akira Nonoyama Santen Pharmaceutical
More informationTransfection of neural stem cells with Lipofectamine Stem Transfection Reagent in StemPro medium
TRANSFECTION PROTOCOL Lipofectamine Stem Transfection Reagent Transfection of neural stem cells with Lipofectamine Stem Transfection Reagent in StemPro medium NSC media, passaging reagents, and complexation
More informationRNP-IP (Modified Method)-Getting Majority RNA from RNA Binding Protein in the Cytoplasm Fengzhi Liu *
RNP-IP (Modified Method)-Getting Majority RNA from RNA Binding Protein in the Cytoplasm Fengzhi Liu * School of Biomedical Sciences, Thomas Jefferson University, Philadelphia, USA *For correspondence:
More informationProtocols for Neural Progenitor Cell Expansion and Dopaminergic Neuron Differentiation
Protocols for Neural Progenitor Cell Expansion and Dopaminergic Neuron Differentiation In vitro neurological research presents many challenges due to the difficulty in establishing high-yield neuronal
More informationHuman Tenocyte Care Manual
Human Tenocyte Care Manual INSTRUCTION MANUAL SHIPPING CONDITIONS Human Tenocytes ZBM0075.03 Orders are delivered via Federal Express courier. Cryopreserved cells are shipped on dry ice and should be stored
More informationHuman IL-6 ELISA Set
Human IL-6 ELISA Set Catalog No. CDK082B Quantity: 10 x 96 tests PRODUCT SPECIFICATIONS : Specificity: Recognizes both natural and recombinant human IL-6 Range: 6.25 pg / ml - 200 pg / ml Sensitivity:
More informationBarbier. European Synchrotron Radiation Facility (ESRF), Grenoble, France. For correspondence:
Manganese Cytotoxicity Assay on Hippocampal Neuronal Cell Culture Alexia Daoust 1, 2, Yasmina Saoudi 1, 2, Jacques Brocard 1, 2, Nora Collomb 1, 2, Cécile Batandier 3, Mariano Bisbal 1, 2, Murielle Salomé
More informationDeliverX and DeliverX Plus sirna Transfection Kits
DeliverX and DeliverX Plus sirna Transfection Kits User Manual P/N 10586 Rev. C 06.09.16 DRAFT September 16, 2006 3:54 pm DeliverXTtile.fm Panomics, Inc. DeliverX and DeliverX Plus sirna Transfection Kit
More informationicell DopaNeurons Kit
icell DopaNeurons Application Protocol Measuring Synchronous Neuronal Activity on the Maestro Multielectrode Array Introduction icell DopaNeurons, human induced pluripotent stem cell (ipsc)-derived neurons,
More informationAlphaScreen SureFire STAT3 Total Assay Kits. Manual
AlphaScreen SureFire STAT3 Total Assay Kits Manual Assay Points Catalog # 500 TGRTS3S500 10 000 TGRTS3S10K 50 000 TGRTS3S50K For Research Use Only Research Reagents for Research Purposes Only TGRKVS73.5
More informationMaterials and Reagents
Isolation of CD34 + Cells from Human Fetal Liver and Cord Blood Institute of Molecular and Cell Biology, 138673, Singapore Qingfeng Chen 1* and Jianzhu Chen 2* 1 Humanized Mouse Unit, Institute of Molecular
More informationData Sheet CD137/NF-κB Reporter - HEK293 Recombinant Cell Line Catalog # 79289
Data Sheet CD137/NF-κB Reporter - HEK293 Recombinant Cell Line Catalog # 79289 Background Human CD137 (4-1BB; TNFRS9) is an inducible co-stimulatory molecule that activates T cells. CD137:CD137L-mediated
More informationGuidelines for WTC Derived AICS hipsc Lines Scale Up & Banking (upto 80 vials)
Version 1.2 2017 Allen Institute for Cell Science Guidelines for WTC Derived AICS hipsc Lines Scale Up & Banking (upto 80 vials) Note: The following protocol is for banking ~60-80 vials of cells (1 million/vial)
More informationab Cellular Reactive Detection Assay Kit (Red Fluorescence) Version 3c Last updated 24 August 2017
Version 3c Last updated 24 August 2017 ab186027 Cellular Reactive Oxygen Species Detection Assay Kit (Red Fluorescence) For the detection of Reactive Oxygen Species (ROS) in live cells. This product is
More informationProduct datasheet. Note: This protocol was modified as of March All previous protocols are obsolete.
Product datasheet Note: This protocol was modified as of March 2017. All previous protocols are obsolete. Mouse total IL-12 (p40 + p70) ELISA Kit Product #: 0006 Storage recommendations Store the kit at
More informationEUCOMM Protocol for ES cells
EUCOMM Protocol for ES cells 1. SNL Feeder Cells 1.1. Preparing inactivated SNL Feeder Cells 1) Thaw one vial of SNL cells (approximately1.5-2 x 10 6 cells per vial) in a 37 C water bath and dilute into
More informationElectroporation of Cas9/Synthetic RNA Ribonucleoprotein (RNP) Complexes for CRISPR/Cas9 Genome Editing (Thermo Neon System)
Electroporation of Cas9/Synthetic RNA Ribonucleoprotein (RNP) Complexes for CRISPR/Cas9 Genome Editing (Thermo Neon System) BACKGROUND This protocol describes how to transfect cultured cells with ribonucleoprotein
More informationAlphaScreen SureFire IκBα Total Assay Kits. Manual
AlphaScreen SureFire IκBα Total Assay Kits Manual Assay Points Catalog # 500 TGRTIKS500 10 000 TGRTIKS10K 50 000 TGRTIKS50K For Research Use Only Research Reagents for Research Purposes Only TGRKVS71.5
More informationBovine IgG-Ab ELISA Kit
Bovine IgG-Ab ELISA Kit For the quantitative in vitro determination of Bovine Immunoglobulin G Antibody concentrations in serum - plasma - tissue homogenates - other biological fluids FOR LABORATORY RESEARCH
More informationhuman Dopamine D 2L Receptor, Frozen Cells
TECHNICAL DATA SHEET AequoZen Caution: For Laboratory Use. A research reagent for research purposes only. You are authorized to utilize these frozen cell preparations one time only. Any attempt to transfer,
More informationCell Line Nucleofector Kit V
page 1 of 7 Cell Line Nucleofector Kit V for THP-1 Cells [ATCC] Cell type Origin Human accute monocyte leukemia cell line [ATCC TIB-202 frozen vial]. Morphology Monocyte % transfection efficiency 80 70
More informationTaqman TCID50 for AAV8 RSM Infectious Titer Determination
Page 1 of 8 Purpose: To determine the concentration of infectious particles in the AAV8 reference standard material. This process involves serial dilution of the vector in a TCID50 format and endpoint
More informationGlobal Histone H4 Acetylation Assay Kit
Global Histone H4 Acetylation Assay Kit Catalog Number KA0634 96 assays Version: 05 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle
More informationHippocampal Neuron Dissociation Transfection and Culture in Microfluidics Chambers Yang Geng *
Hippocampal Neuron Dissociation Transfection and Culture in Microfluidics Chambers Yang Geng * Department of Pediatrics and Bioengineering, Stanford University School of Medicine, Stanford, USA *For correspondence:
More informationHuman Muscarinic M2 Receptor, -Irradiated Frozen Cells
TECHNICAL DATA SHEET AequoZen Caution: For Laboratory Use. A research reagent for research purposes only Human Muscarinic M2 Receptor, -Irradiated Frozen Cells Product No.: ES-211-AF Lot No.: 639-110-A
More informationncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions
ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions with Universal Cell Capture Kit Intracellular Compatible User Manual NanoString Technologies, Inc. 530 Fairview Ave North
More informationTransIT -Lenti Transfection Reagent
Quick Reference Protocol, SDS and Certificate of Analysis available at mirusbio.com/6600 INTRODUCTION Lentivirus is an enveloped, single-stranded RNA virus from the Retroviridae family capable of infecting
More informationEZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK
EZ-0 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK (Bacteria, Plant, Animal, Blood) Version 8 Rev 05/0/03 EZ-0 Genomic DNA Kit Handbook Table of Contents Introduction Limitations of Use Features Applications
More informationTransfection Reagent SPECIFICATIONS MATERIALS. For Research Use Only. Materials Supplied. Materials required, but not supplied
TransIT-siQUEST Transfection Reagent Quick Reference Protocol, MSDS and Certificate of Analysis available at mirusbio.com/2110 INTRODUCTION TransIT-siQUEST is a broad spectrum sirna transfection reagent
More informationInstructions. Fuse-It-siRNA. Shipping and Storage. Overview. Kit Contents. Specifications. Note: Important Guidelines
Membrane fusion is a highly efficient method for transfecting various molecules and particles into mammalian cells, even into sensitive and primary cells. The Fuse-It reagents are cargo-specific liposomal
More informationDeliverX and DeliverX Plus sirna Transfection Kits
DeliverX and DeliverX Plus sirna Transfection Kits User Manual P/N 10586.060504 Rev. A DRAFT May 4, 2006 4:41 pm DeliverXTtile.fm Panomics, Inc. DeliverX and DeliverX Plus sirna Transfection Kit User Manual
More informationMouse OVA IgG1 ELISA. Catalog Number M046072
Mouse OVA IgG1 ELISA Catalog Number M046072 For the quantitative determination of Ovalbumin specific IgG1 in mouse plasma, serum, tissue and cell culture supernate samples. For research use only. This
More informationicell DopaNeurons Application Protocol
icell DopaNeurons Application Protocol Measuring Synchronous Neuronal Activity on the Maestro Multielectrode Array Introduction icell DopaNeurons, human induced pluripotent stem cell (ipsc)-derived neurons,
More informationHuman Hepatic Organoids
Human Hepatic Organoids Product Information Catalog. No. Product Name Format ax41163 Human Hepatic Organoids Stock Conc. Storage on Arrival 1 cryopreserved vial N/A Liquid Nitrogen Thawing Instructions
More informationwith Intracellular-Compatible Universal Cell Capture Kit
MAN-10067-01 with Intracellular-Compatible Universal Cell Capture Kit In this workflow, cells are collected and then bound to the Universal Cell Capture Beads; then the RNA and Protein samples are prepared
More informationVDL106.3 LARGE-SCALE AMPLIFICATION AND PURIFICATION OF ADENOVIRAL VECTOR
1. Purpose 1.1. The purpose of this protocol is to amplify an adenoviral vector from a crude lysate (final product in SOP:VDL105.2 Preparation of Adenoviral Vector Lysate) for production of high titer
More informationData Sheet. Glucocorticoid Receptor Pathway GR-GAL4 Reporter (Luc)-HEK293 cell Line Catalog #: 60655
Data Sheet Glucocorticoid Receptor Pathway GR-GAL4 Reporter (Luc)-HEK293 cell Line Catalog #: 60655 Background The glucocorticoid signaling pathway plays an important role in development, fluid homeostasis,
More informationAvalanche -Everyday Transfection Reagent
The Transfection & Gene Expression Experts Avalanche -Everyday Transfection Reagent Cat. No. EZT-EVDY-1 Description Size: 0.75 ml 1.5 ml Store at 4 C As the simplified version of our most popular and powerful
More informationab CytoPainter MitoBlue Indicator Reagent
Version 1 Last updated 27 April 2017 ab219940 CytoPainter MitoBlue Indicator Reagent For staining mitochondria in live cells using our proprietary MitoBlue Indicator fluorescence probe This product is
More informationThe RNAi Consortium (TRC) Broad Institute
TRC Laboratory Protocols Protocol Title: Lentivirus production of shrna or ORF-pLX clones Current Revision Date: 10/20/2012 RNAi Platform,, trc_info@broadinstitute.org Brief Description: This protocol
More information